Journal of Applied Pharmaceutical Science 02 (08); 2012: 165-170 ISSN: 2231-3354 Micro-morphology of Achillea clypeolata: Received on: 12-07-2012 Revised on: 12-08-2012 Accepted on: 17-08-2011 contribution to the pharmacognostical profile DOI: 10.7324/JAPS.2012.2828 Anely Nedelcheva ABSTRACT Achillea clypeolata Sibth. & Sm. is Balkan endemic species, commonly known as yellow yarrow and traditionally used in Bulgarian folk medicine. The purpose of this study was to establish pharmacognostical profile of A. clypeolata, including macroscopic and microscopic morphological characteristics. The study also aims to propose a method for micromorphological investigation and pharmacognostical identification. As a result were established morphological characters necessary for identification of Achilleae clypeolatae flos and Achilleae clypeolatae herba – morphology of stem leaves (lanceolate in shape, pinnately lobed-margin, acute-apex Anely Nedelcheva Department of Botany with lamina), ligule petals (2.7-3 mm), involucre bracts (4.5x2 mm) and achenes (1.5-1.7 mm); Sofia University “St. Kliment micromorphology of lamina (110 -130 µm), epidermal cells, stomata, glandular, non-glandular Ohridski”, Faculty of Biology trichomes and 3-colporate, echinate pollen grains (20.5-20.8 µm). This study presents new blvd. Dragan Tzankov 8, Sofia–1164 micromorphological data. The used staining method dyed various tissues; makes visible the Bulgaria outlines of structures, tissues and some inclusions; allows by one procedure to visualize important diagnostic features; unlike many other methods, allowing pollen to be used as a characteristic of herbal substances. The results are useful for quality control methods for medicinal plant materials and contributes to the implementation of good practices for plant identification for the herbal industry, food, nutrient supplements and other plant based products. Keywords: Yellow yarrow, Herba, Flos, Pharmacognosy, Balkan endemic, Bulgaria. INTRODUCTION Genus Achillea L. is one of the most polymorphic genera and difficult for interpretation in the family Asteraceae and is object of recent taxonomical comments on the basis of multidisciplinary studies (Ehrendorfer and Guo, 2005). Biosystematic studies of Achillea have a tradition of long standing in Bulgarian botany (Kuzmanov, 1984, Nedelcheva, 1998, Saukel et al., 2003). According to the latest data, the genus comprises about 20 (3+17) species of perennial herbs distributed in Bulgaria, belonging to two sections: sect. Anthemoideae (DC.) Heimerl s.l. and sect. Achillea s.l. [= sect. Millefolium (Adanson) Koch s.l., incl. Sect. Filipendulinae (DC.) Afan.]. For Correspondence In Bulgaria are known 6 species with yellow flowers, (A. clypeolata Sibth. & Sm., A. coarctata Anely Nedelcheva Poir., A. pseudopectinata Janka s.lat., A. chrysocoma Friv., A. thracica Velen. and A. ochroleuca Phone: (+359 2) 8167 /304/ [email protected] Ehrh.) (Saukel et al., 2003, Nedelcheva and Tzonev, 2006). Email: Journal of Applied Pharmaceutical Science 02 (08); 2012: 165-170 Achillea clypeolata is a Balkan endemic species and MATERIALS AND METHODS ranges from North and Central Greece to South Albania, Preparation of the plant material and samples Macedonia, Serbia, Bulgaria, South-east Romania, West and Aboveground parts of the plant are used herbal substance: European Turkey. Together with Achillea aegyptiaca L. (incl. Achilleae clypeolatae flos and Achilleae clypeolatae herba Achillea taygetea Boiss.) they form the dominantly diploid and (Nikolov, 2006). The present study is examination of the dry and Balkan centered group of A. clypeolata, characterized by grounded plant material collected from natural areas and morphometric and phytochemical features. A. clypeolata is most commercial herb samples (product contains dried herbs) (Table 1). morphological close to Achillea coarctata (Richardson, 1976, Medium representative sampling were separated and processed. Saukel et al., 2003). Warm water (up to 30ºC) is poured over a certain amount of dried Among species with yellow flowers, mostly A. clypeolata herb, covered, and allowed to steep for 20-40 minutes. For commonly known as “yellow yarrow” and traditionally used in temporary or permanent microscope observation small amount of Bulgarian folk medicine to treat hemorrhoids, wounds, bleeding, wet herbal fragments were placed on microscope slides and mixed gastro-intestinal atony, bed wetting, kidney inflammation, with 1-2 drops from differential staining mixture and fixed with amenorrhoea and inflamed gums, liver deseases. Its medicinal cover slips. The cover-slips of slides intended for storage were effect is like white yarrow, which is more widespread and better sealed with paraffin wax to prevent excessive evaporation of the known in folk medicine. Bulgarian folk names: “zhult ravnets”, mounting medium. The mounts were kept in laboratory oven at “strumski ravnets”, “div pelin”, “zhulta eniovitsa”, “skalen 50ºC for 24 hr. Mounts stored for over a 6 months (refrigerator 2- ravnets”, “ramchets”, “glavoch” and “gushterka”. The large 6ºC) showed no loss of staining. number and variety of folk names showed that the plant is well known and wide used. They are based mainly on its yellow color, Table 1: List of the specimens used in morphological investigations. shape of the inflorescence, bitter taste like mugwort and rocky Natural areas Commercial herbal samples habitats (Ahtarov et al., 1939; Nedelcheva and Dogan, 2009). herbal samples Some of the current research focuses on the AN/1/645; AN/1/304; Bilki Eood/ 2010 pharmacological properties of the yellow yarrow (Konyalioglu and Achilleae clypeolatae AN/1/077; Karmen/2005 flos Karamenderes, 2005; Karaalp et al., 2009). Significantly more are AN/1/532; Suni Yambol EOOD/2010 the data about white yarrow as herbal drug (herbal substance) AN/1/062 (Rauchensteiner et al., 2004; Nirmala and Karthiyayini, 2010). AN/2/005; AN/2/112; Of central importance in the practice is correct Achilleae clypeolatae Bilki Eood/ 2010; Karmen AN/2/452; herba Solara /2009 identification of the species concerned, whether in the fresh, dried AN/2/33; or powdered state, because one of the criticisms of herbal medicine AN/2/076 is lack of standardization and quality control profiles. The macroscopic and microscopic description of a medicinal plant is The stain was prepared according to Alexander (1969) the first step towards establishing the identity and the degree of from various constituents: malachite green, glycerol, phenol, purity of such materials (identity tests) and should be carried out chloral hydrate, acid fuchsin, orange G, glacial acetic acid, before any tests to be undertaken (WHO, 1998). Therefore, more glycerol, 95% alcohol, distilled water. The amount of glacial acetic nowadays studies are conducted on micromorhology of herbal acid added to mixture is (4 ml to 100 ml stain mixture), because drugs for their correct identification (Jayeola, 2009; HMPC, 2010; thick-walled and spiny pollen grains as well as 5 mg acid fuchsin HMPC, 2010a). For A. clypeolata there is no pharmacopoeial additionally with aim to improve contrast (Nedelcheva, 1998). In monograph and in control of starting herbal material (including the staining process malachite green is used for staining cell walls mother plant identification) should be followed detailed and pollen walls. specification of the quality controls (EDQM, 2011; Nikam et al., Acid fuchsin in the present mixture is included for 2012). The wide use of the yellow yarrow as a herb in Bulgaria, its staining the protoplasm and also colored non aborted pollen grains place in folk medicine and its use today in the herbal remedies, in red to red-purple. These characteristics gave reason to along with limited data as a plant substance justify the purpose of experiment with this stain solution and on plant tissues, not only to this study: to establish pharmacognostical profile of A. clypeolata, differential staining of aborted and non aborted pollen grains which including macroscopic and microscopic morphological was the target of Alexander (1969). characteristics. The study also aims to propose a method for For morphological investigations related size measures, micromorphological investigation and pharmacognostical twenty plant individuals from each population or samples were identification of the herbal substances which is informative, fast studied. Dry herbal substances were analyzed and photographed and with easy laboratory performance as well as applicable in the using the light microscope Olympus BX with digital camera system of herbal gathering and herb production, preparation and Olympus SP-510UZ. implementation of marketing, quality control of herbal Voucher specimens was deposited in the personal author preparations, food, nutrient supplements and other plant based collection in Department of Botany and in the Herbarium of products. University of Sofia (SO). Journal of Applied Pharmaceutical Science 02 (08); 2012: 165-170 Study area Based on this preliminary analysis was performed Bulgaria (42°41′0″N, 23°19′0″E) is a country in the micromorphological investigation on most involved characteristics Balkans in south-eastern Europe. It borders five other countries: with diagnostic value and last but not least susceptible to dying by Romana to the north (mostly along the River Danube), Serbia and the proposed method. Inflorescences are yellow, dense, flat-topped the Republic of Macedonia to the west, and Greece and Turkey to and 3-5(7) сm wide corymbs. They